BACKGROUND: Gliomas display cellular hierarchies with tumor initiating cells (TICs) at the apex that are functionally defined by the ability to self renew and propagate tumors similar to the parental tumors from which they are derived. TICs remain controversial, but their clinical relevance has been supported by resistance to cytotoxic therapies (Bao et al. Nature 2006) and promotion of tumor angiogenesis (Bao et al. Cancer Research 2006). TICs reside in specific functional niches in perivascular and hypoxic niches (Li et al. Cancer Cell 2009) that may offer the ability to disrupt tumor maintenance and therapeutic resistance through targeting the niche. Investigating TICs has already yielded novel molecular targets and pathways that are amenable to therapeutic targeting (Kim et al. Genes Development 2012; Eyler et al. Cell 2011; Guryanova et al. Cancer Cell 2011). METHODS: Using patient-derived tumor models, we interrogated the regulation of the TIC phenotype by both cell intrinsic and microenvironmental influences present in tumors. RESULTS: TICs are enriched under low nutrient conditions due to the cooption of the high affinity GLUT3 transporter normally expressed by neurons (Flavahan et al. Nature Neuroscience 2013). We have now extended these findings to demonstrate that cellular metabolism is differentially regulated within the tumor hierarchy at several levels to provide resources for sustained self-renewal and proliferation. We also recently found that TICs have basal genotoxic stress activating PARP permitting radiosensitization (Venere et al. Cell Death Differentiation 2014). To discover novel TIC targets, we are using several technologies, including aptamers (Kim et al. Cancer Research 2013), flow cytometry (Lathia et al. Cell Reports 2014), and in unpublished studies, phage display, showing that TICs manifest nodes of fragility mediating cell survival and invasion (e.g. JAM-A, VAV3, and CD97). Combining TIC models from patients with non-neoplastic progenitors from epilepsy resections, we are interrogating additional molecular regulators of the cellular hierarchy that can be distinguished from normal stem cells to minimize toxicity. CONCLUSIONS: The conventional pyramidal unidirectional differentiation cascade with TICs at the apex has been called into question by studies demonstrating plasticity of the TIC phenotype (Cheng et al. Cell 2013), thus suggesting that targeting only TICs will likely fail to cure patients and require simultaneous targeting of TICs and the bulk tumor. Although the field of TIC biology is relatively young, continued elucidation of the tumor hierarchy holds promise for development of novel patient therapies. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.